Hepatitis B virus (HBV) infection is a significant health burden worldwide. Over 350 million individuals are chronically infected with HBV and ~600,000 deaths annually are attributed to HBV-related liver disease. It is the leading cause of hepatocellular carcinoma worldwide. Seven therapies for HBV are available in the clinic but none are a cure. A better understanding of the HBV life cycle on the molecular and cellular level will lead to new therapies for HBV. The capsid is an attractive target for therapy because of its central role in virus replication. Beyond its well-characterized role as a structural component of the virus, the capsid is a dynamic entity that plays roles in multiple processes such as genome replication, its intracellular trafficking, cccDNA synthesis, and virus morphogenesis. Project 2 seeks to establish new understandings of the mechanisms of these roles of the HBV capsid. This present proposal builds upon progress engendered during the current funding period. This project has four aims: 1) to understand how perturbation of the capsid dimer-dimer interface impacts the HBV life cycle; 2) to visualize HBV assembly and replication using live cell imaging; 3) to better understand the relationship between DNA maturation, capsid maturation and virion secretion using avian hepadnaviruses; and 4) to use hepatocyte-like cells derived from embryonic stem cells to study early events in HBV infection. These studies will uncover new understandings of mechanisms of virus replication and identify targets for new and better HBV therapies. Project 2 is dependent on productive interactions with Project 3 for knowledge and expertise on live cell imaging. Aim 4 has the potential for important interactions with Project 5 in the use of mice with humanized immune systems to study HBV:host interactions. Current research findings from Project 2 have influenced new research directions in Project 1 with regard to ZASCI's role in cervical carcinogenesis. Project 2 relies heavily on all three cores, including all instruments supported by the Instrument Core, HBV production capabilities of the Virus Core, and statistical analysis of experimental design and results from the Admin./Stats Core.